The development of gene therapy for hemophilia and other diseases remains stymied by a failure to achieve clinically significant levels of transgene integration in targeted cells. Adeno-Associated Virus integrates into Human Chromosome 19qter and increasing evidence suggests a role for the viral Rep protein in the integration process. The purpose of this project is to understand the role of the adeno- associated virus (AAV) Rep protein in AAV integration and incorporate this property into a non-viral vector system designed to achieve targeted integration of heterologous transgenes flanked by AAV ITRs (Inverted Terminal Repeats; the AAV Origins of Replication). We made significant progress in this work in the past year. Using a novel, liposome-based system, a marker gene flanked by AAV ITR's was delivered to human hepatoma G2 cells. When MBP-Rep protein was also delivered, there was a Rep dose-dependent increase in the percentage of cells expressing the marker gene two months following the initial transfection. These results are consistent with a role for the AAV Rep protein in integration. We are extending these experiments by developing a cell line transgenic for AAVS1, the Human Chromosome 19qter locus into which AAV integrates. Our ultimate goal is to develop a non- viral gene therapy system which achieves in vivo integration of genes for Human Coagulation Factors VIII and IX.